Compositions and methods of use of phorbol esters for the treatment of stroke

ABSTRACT

Methods and compositions containing a phorbol ester or a derivative of a phorbol ester are provided for the treatment and prevention of stroke and the sequelae of stroke. Additional compositions and methods are provided which employ a phorbol ester or derivative compound in combination with at least one additional agent to yield more effective treatment tools to treat or prevent stroke and the long term effects of stroke in mammalian subjects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/358,388, filed Nov. 22, 2016 (now allowed), which is a Continuationof U.S. application Ser. No. 14/026,534, filed Sep. 13, 2013 (now U.S.Pat. No. 9,533,938 issued Jan. 3, 2017); which is a Continuation-In-Partof (1) U.S. application Ser. No. 13/745,742, filed Jan. 18, 2013; whichclaims priority benefit of U.S. Provisional Patent Application Ser. No.61/588,167, filed Jan. 18, 2012 (now expired); and (2) U.S. applicationSer. No. 13/794,467, filed Mar. 11, 2013 (now U.S. Pat. No. 9,132,113issued Sep. 15, 2015), which is a Continuation of U.S. application Ser.No. 13/595,072, filed Aug. 27, 2012 (now abandoned), which is aContinuation of U.S. application Ser. No. 12/023,753, filed Jan. 31,2008 (now abandoned), which claims priority benefit of U.S. ProvisionalPatent Application Ser. No. 60/898,810, filed Jan. 31, 2007 (nowexpired); the contents of each of which are incorporated herein byreference in their entireties.

ADDITIONAL DISCLOSURE

Additional disclosures relating to the instant application may be foundin “Compositions And Methods Of Use Of Phorbol Esters” U.S. patentapplication Ser. No. 12/023,753, filed Jan. 31, 2008, to Richard L.Chang, et al, which claims priority benefit of U.S. Provisional PatentApplication Ser. No. 60/898,810, filed Jan. 31, 2007, each of which isincorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

The present invention relates generally to the medicinal use of phorbolesters in the treatment and prevention of stroke and the effects ofstroke.

BACKGROUND

Plants have historically served many medicinal purposes. The WorldHealth Organization (WHO) estimates that 4 billion people, 80% of theworld's population, presently use herbal medicine for some aspect ofprimary health care. (WHO Fact sheet Fact sheet N^(o) 134, December2008) However, it can be difficult to isolate the specific compound in aplant that has the desired medicinal effect and to reproduce it on acommercial scale. Additionally, while the active compound may beisolated from a plant, the other parts of a plant such as the minerals,vitamins, volatile oils, glycosides, alkaloids, bioflavanoids, and othersubstances may also be involved in the functioning of the activecompound or the medicinal effect for which the plant is known, makingthe use, purification and commercialization of plant basedpharmaceutical agents a challenge.

Phorbol is a natural, plant-derived organic compound of the tiglianefamily of diterpenes. It was first isolated in 1934 as a hydrolysisproduct of croton oil derived from the seeds of Croton tiglium, a leafyshrub of the Euphorbiaceae family that is native to Southeastern Asia.Various esters of phorbol have important biological properties includingthe reported ability to mimic diacylglycerols and activate proteinkinase C (PKC); and to modulate downstream cell signaling pathwaysincluding the mitogen-activated protein kinase (MAPK) pathways. Phorbolesters are additionally thought to bind to chimaerins, the Ras activatorRasGRP, and the vesicle-priming protein Munc-13 (Brose N, Rosenmund C.,J Cell Sci; 115:4399-411 (2002)). Some phorbol esters also inducenuclear factor-kappa B (NF-κB). The most notable physiological propertyof phorbol esters is their reported capacity to act as tumor promoters.(Blumberg, 1988; Goel, G et al., Int, Journal of Toxicology 26, 279-288(2007)).

12-O-tetradecanoylphorbol-13-acetate (TPA), also calledphorbol-12-myristate-13-acetate (PMA), is a phorbol ester used in modelsof carcinogenesis as an inducer for differentiation and/or apoptosis inmultiple cell lines and primary cells. TPA has also been reported tocause an increase in circulating white blood cells and neutrophils inpatients whose bone marrow function has been depressed by chemotherapy(Han Z. T. et al. Proc. Natl. Acad. Sci. 95, 5363-5365 (1998)), and toinhibit an HIV-induced cytopathic effect on MT-4 cells. (Mekkawy S. etal., Phytochemistry 53, 47-464 (2000)). However, due to a variety offactors, including caustic reactions when contacted with the skin andconcerns for its potential toxicity, TPA has not been shown to be aneffective tool for treating, managing, or preventing disease. Indeed, asphorbol esters play a key role in activation of protein kinase C, whichtriggers various cellular responses resulting in inflammatory responsesand tumor development (Goel et al., Int, Journal of Toxicology 26,279-288 (2007)), phorbol esters would generally be excluded frompossible treatment candidates for conditions that involve inflammatoryreactions such as stroke.

Heart disease and stroke kill some 17 million people a year, causingalmost one-third of all deaths globally. They are predicted to becomethe leading cause of both death and disability worldwide, with thenumber of fatalities projected to increase to over 20 million a year by2020 and to 24 million a year by 2030. (Atlas of Heart Disease andStroke, World Health Organization 2004).

While there are over 300 risk factors associated with coronary heartdisease and stroke (Atlas of Heart Disease and Stroke, World HealthOrganization 2004), in developed countries, at least ⅓ of allcardiovascular disease is attributable to tobacco use, alcohol use, highblood pressure, high cholesterol and obesity.

Current treatments for the management and prevention of stroke aregenerally a combination of medications such as ACE inhibitors, aspirin,beta blockers and lipid lowering medications; devices such aspacemakers, implantable defibrillators, coronary stents, prostheticvalves and artificial hearts; and operations such as coronary arterybypass, balloon angioplasty, valve repair and replacement, hearttransplants and artificial heart operations. “Increasingly,high-technology procedures are chosen over less expensive, butnevertheless effective, strategies” (Atlas of Heart Disease and Stroke,World Health Organization 2004) adding to the rising costs of healthcare and leading to marked disparities in the quality of treatmentbetween different groups of individuals.

However, even where advanced technology and facilities are available,60% of those who suffer a stroke die or become dependent and each strokesignificantly increases the risk of further episodes. “Worldwide,treatment of cardiovascular diseases and their risk factors remainsinadequate for most patients.” (Atlas of Heart Disease and Stroke, WorldHealth Organization 2004). There is therefore clearly a need for new andmore effective measures to treat and prevent strokes and to treat orprevent the long term effects caused by stroke.

SUMMARY

The present invention relates to compositions containing and methods ofusing phorbol esters. The compositions and methods described herein areeffective in preventing and treating stroke and in managing the sequelaeof stroke including acute ischemic events.

Effects of stroke that may be prevented or treated by using the phorbolesters and derivatives of phorbol esters as described herein include,but are not limited to, paralysis, spatial impairment, impairedjudgment, left-sided neglect, memory loss, aphasia, coordination andbalance problems, nausea, vomiting, cognitive impairment, perceptionimpairment, orientation impairment, homonymous hemianopsia andimpulsivity. In some embodiments, the use of phorbol esters andderivatives of phorbol esters may prevent initial and subsequent strokesfrom occurring.

Successful treatment will be determined according to conventionalmethods, such as a reduction in the severity or sequelae of a stroke, adecrease or elimination of the effects of stroke, a decrease in riskfactors that predispose an individual to a stroke, and/or a decrease inthe number or severity of strokes including subsequent strokes.

In another embodiment, the phorbol esters and derivatives of phorbolesters as described herein may be used to modulate cell signalingpathways. Such modulation may have a variety of results, for example, insome embodiments, the use of compositions containing phorbol esters andderivatives of phorbol esters may alter the release of Th1 cytokines inmammalian subjects. In a further embodiment, compositions containingphorbol esters and/or phorbol ester derivatives may alter the release ofinterleukin 2 (IL-2) in mammalian subjects. In an additional embodiment,compositions containing phorbol esters and/or phorbol ester derivativesmay alter the release of interferon in mammalian subjects. In yetanother embodiment, compositions containing phorbol esters and/orphorbol ester derivatives may alter the rate of ERK phosphorylation.

The invention achieves the foregoing and satisfies additional objectsand advantages by providing novel and surprisingly effective methods andcompositions for treating and preventing stroke, modulating cellsignaling pathways and/or managing, treating and preventing the sequelaeof a stroke using compositions containing a phorbol ester of Formula I,below:

wherein R₁ and R₂ may independently be hydrogen; hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein a lower alkenyl group contains between 1 to 7 carbon atoms;

and substituted derivatives thereof. R₃ may be independently hydrogen or

and substituted derivatives thereof. The methods and compositions of thepresent invention further include any pharmaceutical salts, enantiomers,isomer, polymorphs, prodrugs, hydrates and solvates of the compositionsof Formula I.

In some embodiments, at least one of R₁ and R₂ are other than hydrogenand R₃ is hydrogen or

and substituted derivatives thereof. In another embodiment, either R₁ orR₂ is

the remaining R₁ or R₂ is a

wherein a lower alkyl is between 1 and 7 carbons, and R₃ is hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups of the formulas of FormulaI herein may be unsubstituted or substituted with halogens, preferably,chlorine, fluorine or bromine; nitro; amino; and/or similar typeradicals.

An exemplary phorbol ester composition as used in the methods andcompositions as described herein is 12-O-tetradecanoylphorbol-13-acetate(TPA) of Formula II, below:

Useful phorbol esters of Formula I and related compounds and derivativeswithin the formulations and methods of the invention include, but arenot limited to, other pharmaceutically acceptable active salts of saidcompounds, as well as active isomers, enantiomers, polymorphs,glycosylated derivatives, solvates, hydrates, and/or prodrugs of saidcompounds. Exemplary forms of phorbol esters for use within thecompositions and methods of the invention include, but are not limitedto, phorbol 13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate;phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate;phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol12-myristate; phorbol 13-myristate; phorbol 12-myristate-13-acetate(also known as TPA or PMA); phorbol 12,13,20-triacetate; 12-deoxyphorbol13-angelate; 12-deoxyphorbol 13-angelate 20-acetate; 12-deoxyphorbol13-isobutyrate; 12-deoxyphorbol 13-isobutyrate-20-acetate;12-deoxyphorbol 13-phenylacetate; 12-deoxyphorbol 13-phenylacetate20-acetate; 12-deoxyphorbol 13-tetradecanoate; phorbol 12-tigliate13-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; andphorbol 13-acetate.

Mammalian subjects amenable to treatment with phorbol esters of FormulaI or derivatives of a phorbol ester of the Formula I, particularly TPA,according to the methods of the invention include, but are not limitedto, individuals who have suffered or are at risk for a stroke. Subjectsamenable to treatment with phorbol esters of Formula I, particularlyTPA, additionally include those suffering from the effects of a strokeincluding, but not limited to, paralysis, spatial impairment, impairedjudgment, left-sided neglect, memory loss, aphasia, coordination andbalance problems, nausea, vomiting, cognitive impairment, perceptionimpairment, orientation impairment, homonymous hemianopsia andimpulsivity.

These and other subjects are effectively treated prophylactically and/ortherapeutically, by administering to the subject an effective amount ofa phorbol ester of Formula I or derivatives of a phorbol ester of theFormula I sufficient to modulate NF-κB activity, increase Th1 cytokineactivity, prevent or treat paralysis, increase spatial awareness,decrease memory loss, decrease aphasia, increase coordination andbalance, prevent or decrease the incidence and severity of a stroke, andimprove cognition.

Therapeutically useful methods and formulations of the invention willeffectively use a phorbol ester of Formula I or derivative of a phorbolester of the Formula I in a variety of forms, as noted above, includingany active, pharmaceutically acceptable salts of said compounds, as wellas active isomers, enantiomers, polymorphs, solvates, hydrates,prodrugs, and/or combinations thereof. TPA of formula II is employed asan illustrative embodiment of the invention within the examples hereinbelow.

Within additional aspects of the invention, combinatorial formulationsand methods are provided which employ an effective amount of a phorbolester of Formula I or derivative of a phorbol ester of Formula I incombination with one or more secondary or adjunctive active agent(s)that is/are combinatorially formulated or coordinately administered withthe phorbol ester compound of Formula I to yield an effective responsein the subject.

Combinatorial formulations and coordinate treatment methods in thetreatment of stroke employ a phorbol ester compound of Formula I orderivative of the phorbol ester of Formula I in combination with one ormore additional, stroke preventing, treating or other indicated,secondary or adjunctive therapeutic agents. The secondary or adjunctivetherapeutic agents used in combination with a phorbol ester, e.g., TPA,in these embodiments may possess direct or indirect effects onprevention or recovery from stroke, alone or in combination with thephorbol ester, e.g. TPA; may exhibit other useful adjunctive therapeuticactivity in combination with, e.g. TPA (such as anti-clotting,anticholesterolemic, vasodilating, antihypertensive, arteriolarresistance lowering, venous capacity increasing, heart oxygen demandreducing, heart rate decreasing, heart rate stabilizing, orneuroprotecting); or may exhibit adjunctive therapeutic activity usefulfor treating or preventing stroke or associated symptoms alone or incombination with, e.g. TPA. Such secondary or adjunctive therapeuticagents may be administered prior to, simultaneously, or afteradministration of a phorbol ester of Formula I or derivative of aphorbol ester of Formula I.

Useful adjunctive or secondary therapeutic agents in these combinatorialformulations and coordinate treatment methods for the prevention ortreatment of stroke in a mammalian subject include, but are not limitedto, tissue plasminogen activators, anticoagulants, statins, angiotensinII receptor blockers, angiotensin-converting enzyme inhibitors,anti-platelet agents, beta-blockers, aspirin, fibrates, calcium channelblockers, or diuretics. In addition, adjunctive or secondary therapiesmay be used such as, but not limited to, surgical intervention includingcarotid endarterectomy, angioplasty, balloon angioplasty, valve repairand replacement, coronary artery bypass, stent placement, craniotomy,endovascular coil emobilization, or patent foramen ovale closure.

The foregoing and additional objects, features, aspects and advantagesof the present invention will become apparent from the followingdetailed description.

DETAILED DESCRIPTION

Novel methods and compositions have been identified for use inpreventing and/or treating stroke and the sequelae of stroke inmammalian subjects, including humans.

In various embodiments, the composition and methods as described hereinmay increase the release of Th1 cytokines, increase ERK phosphorylation,modulate NF-κB activity, prevent or treat paralysis, increase spatialawareness, decrease memory loss, decrease aphasia, increase coordinationand balance, improve cognition, improve orientation, decrease theprevalence of subsequent strokes, and decrease impulsivity.

Formulations and methods provided herein employ a phorbol ester ofFormula I or derivative compound of a phorbol ester of Formula I as morefully described in U.S. patent application Ser. No. 12/023,753, filedJan. 31, 2008, which claims priority benefit of U.S. Provisional patentapplication Ser. No. 60/898,810, filed Jan. 31, 2007 which isincorporated herein in its entirety by reference,

wherein R₁ and R₂ may independently be hydrogen; hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein a lower alkenyl group contains between 1 to 7 carbon atoms;

and substituted derivatives thereof. R₃ may be independently be hydrogenor

In some embodiments, at least one of R₁ and R₂ are other than hydrogenand R₃ is hydrogen or

and substituted derivatives thereof as novel compositions for use intreating chronic or recurring conditions. In another embodiment, eitherR₁ or R₂ is

the remaining R₁ or R₂ is a

wherein a lower alkyl is between 1 and 7 carbons, and R₃ is hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein maybe unsubstituted or substituted with halogens, preferably, chlorine,fluorine or bromine; nitro; amino; and/or similar type radicals.

Stroke treating formulations and methods provided herein employ aphorbol ester of Formula I or derivative compound of a phorbol ester ofFormula I, above, including all active pharmaceutically acceptablecompounds of this description as well as various foreseen and readilyprovided complexes, salts, solvates, isomers, enantiomers, polymorphsand prodrugs of these compounds and combinations thereof as anti-strokeagents.

Th1 cytokine increasing formulations and methods provided herein employa phorbol ester or derivative compound of Formula I, above, includingall active pharmaceutically acceptable compounds of this description aswell as various foreseen and readily provided complexes, salts,solvates, isomers, enantiomers, polymorphs and prodrugs of thesecompounds and combinations thereof as novel Th1 cytokine increasingagents. A broad range of mammalian subjects, including human subjects,are amenable to treatment using the formulations and methods of theinvention. These subjects include, but are not limited to, individualswho have suffered or are at risk for a stroke.

ERK phosphorylation increasing formulations and methods provided hereinemploy a phorbol ester or derivative compound of Formula I, above,including all active pharmaceutically acceptable compounds of thisdescription as well as various foreseen and readily provided complexes,salts, solvates, isomers, enantiomers, polymorphs and prodrugs of thesecompounds and combinations thereof as novel ERK phosphorylationincreasing agents. A broad range of mammalian subjects, including humansubjects, are amenable to treatment using the formulations and methodsof the invention. These subjects include, but are not limited to,individuals who have suffered or are at risk for a stroke.

Within the methods and compositions of the invention, one or morephorbol ester compound(s) of Formula I or derivative compounds of aphorbol ester of Formula I as disclosed herein is/are effectivelyformulated or administered as an agent effective for treating andpreventing stroke or the sequelae of stroke. In exemplary embodiments,TPA is demonstrated for illustrative purposes to be an effective agentin pharmaceutical formulations and therapeutic methods, alone or incombination with one or more adjunctive therapeutic agent(s). Thepresent disclosure further provides additional, pharmaceuticallyacceptable phorbol ester compounds in the form of a native or syntheticcompound, including complexes, derivatives, salts, solvates, isomers,enantiomers, polymorphs, and prodrugs of the compounds disclosed herein,and combinations thereof, which are effective as therapeutic agentswithin the methods and compositions of the invention in the treatmentand prevention of stroke and the sequelae of stroke.

Strokes are caused by disruption of the blood supply to the brain. Thismay result from either blockage (ischaemic stroke) or rupture of a bloodvessel (haemorrhagic stroke). The symptoms of a stroke include suddennumbness or weakness, especially on one side of the body; suddenconfusion or trouble speaking or understanding speech; sudden troubleseeing in one or both eyes; sudden trouble with walking, dizziness, orloss of balance or coordination; or sudden severe headache with no knowncause. Risk factors for stroke include high blood pressure, abnormalblood lipids, tobacco use, physical inactivity, obesity, stress,diabetes, alcohol use, excess homocystein in the blood, inflammation andabnormal coagulation. There are also non-modifiable risk factors such asage, heredity, gender and ethnicity.

Treatment for stroke has three distinct phases: prevention, therapyimmediately after a stroke, and post-stroke rehabilitation. Thecompositions and methods described herein may be used at any phase ofstroke treatment, independently or in conjunction with one or moreadditional therapies including other pharmaceutical agents, devices orsurgical interventions.

Phorbol is a natural, plant-derived polycyclic alcohol of the tiglianefamily of diterpenes. It was first isolated in 1934 as the hydrolysisproduct of croton oil derived from the seeds of Croton tiglium. It iswell soluble in most polar organic solvents and in water. Esters ofphorbol have the general structure of Formula I, below:

wherein R₁ and R₂ are selected from the group consisting of hydrogen;hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein a lower alkenyl group contains between 1 to 7 carbon atoms;

and substituted derivatives thereof. R₃ may be hydrogen or

and substituted derivatives thereof as well as pharmaceuticallyacceptable salts, enantiomers, polymorphs, prodrugs solvates andhydrates of compounds of Formula I.

The term “lower alkyl” or “lower alkenyl” as used herein means moietiescontaining 1 to 7 carbon atoms. In the compounds of the Formula I, thealkyl or alkenyl groups may be straight or branched chain. In someembodiments, either or both R₁ or R₂, are a long chain carbon moiety(i.e., Formula I is decanoate or myristate).

The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein maybe unsubstituted or substituted with halogens, preferably, chlorine,fluorine or bromine; nitro; amino and similar type radicals.

Organic and synthetic forms of phorbol esters, including anypreparations or extracts from herbal sources such as croron tiglium, arecontemplated as useful compositions comprising phorbol esters (orphorbol ester analogs, related compounds and/or derivatives) for usewithin the embodiments herein. Useful phorbol esters and/or relatedcompounds for use within the embodiments herein will typically have astructure as illustrated in Formula I, although functionally equivalentanalogs, complexes, conjugates, and derivatives of such compounds willalso be appreciated by those skilled in the art as within the scope ofthe invention.

In more detailed embodiments, illustrative structural modificationsaccording to Formula I above will be selected to provide usefulcandidate compounds for treating and/or preventing strokes, damage dueto stroke and/or managing the effects or sequelae of strokes inmammalian subjects, including humans, wherein: at least one of R₁ and R₂are other than hydrogen and R₃ is selected from the group consisting ofhydrogen or

and substituted derivatives thereof. In another embodiment, either R₁ orR₂ is

the remaining R₁ or R₂ is

and R₃ is hydrogen.

An exemplary embodiment of a phorbol ester compound of Formula I usefulin the treatment of in treating or preventing strokes, damage fromstrokes, and/or managing the effects or sequelae of strokes in mammaliansubjects, including humans, is found in phorbol 12-myristate-13-acetate(also known as PMA or 12-O-tetradecanoyl-phorbol-13-acetate (TPA)) shownin Formula II, below.

Additional useful phorbol esters and related compounds and derivativeswithin the formulations and methods of the invention include, but arenot limited to, other pharmaceutically acceptable active salts of saidcompounds, as well as active isomers, enantiomers, polymorphs,glycosylated derivatives, solvates, hydrates, and/or prodrugs of saidcompounds. Derivatives of phorbol esters of Formula I may or may not bephorbol esters themselves. Further exemplary forms of phorbol esters foruse within the compositions and methods of the invention include, butare not limited to, phorbol 13-butyrate; phorbol 12-decanoate; phorbol13-decanoate; phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate;phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol12-myristate; phorbol 13-myristate; phorbol 12,13,20-triacetate;12-deoxyphorbol 13-angelate; 12-deoxyphorbol 13-angelate 20-acetate;12-deoxyphorbol 13-isobutyrate; 12-deoxyphorbol13-isobutyrate-20-acetate; 12-deoxyphorbol 13-phenylacetate;12-deoxyphorbol 13-phenylacetate 20-acetate; 12-deoxyphorbol13-tetradecanoate; phorbol 12-tigliate 13-decanoate; 12-deoxyphorbol13-acetate; phorbol 12-acetate; and phorbol 13-acetate as shown in Table1.

TABLE 1 Exemplary Phorbol Esters Phorbol 13-Butyrate

Phorbol 12- Decanoate

Phorbol 13- Decanoate

Phorbol 12,13- Diacetate

Phorbol 13,20- Diacetate

Phorbol 12,13- Dibenzoate

Phorbol 12,13- Dibutyrate

Phorbol 12,13- Didecanoate

Phorbol 12,13- Dihexanoate

Phorbol 12,13- Dipropionate

Phorbol 12-Myristate

Phorbol 13-Myristate

Phorbol 12- Myristate-13-Acetate (also known as TPA or PMA)

Phorbol 12,13,20- Triacetate

12-Deoxyphorbol 13-Angelate

12-Deoxyphorbol 13-Angelate 20- Acetate

12-Deoxyphorbol 13-Isobutyrate

12-Deoxyphorbol 13-Isobutyrate-20- Acetate

12-Deoxyphorbol 13-Phenylacetate

12-Deoxyphorbol 13-Phenylacetate 20- Acetate

12-Deoxyphorbol 13-Tetradecanoate

Phorbol 12-Tigliate 13-Decanoate

12-Deoxyphorbol 13-Acetate

Phorbol 12-Acetate

Phorbol 13-Acetate

Compositions as described herein comprise stroke treating compositionscomprising a stroke damage alleviating or preventing effective amount ofa phorbol ester compound of Formula I or derivative compound of aphorbol esters of Formula I, which is effective for prophylaxis and/ortreatment of stroke or stroke related symptoms or sequelae in amammalian subject. A “stroke treating,” “anti-clotting,”“anticholesterolemic,” “vasodilating,” “antihypertensive,” “arteriolarresistance lowering,” “venous capacity increasing,” “heart oxygen demandreducing,” “heart rate decreasing,” “heart rate stabilizing,” or“neuroprotective” effective amount of the active compound istherapeutically effective, in a single or multiple unit dosage form,over a specified period of therapeutic intervention, to measurablyalleviate one or more symptoms or sequelae of stroke in the subject.Within exemplary embodiments, the compositions of the invention areeffective in treatment methods to prevent or alleviate symptoms ofstroke or sequelae of stroke in human and other mammalian subjectsvulnerable to or who have suffered a stroke.

Phorbol ester treating compositions of the invention typically comprisean effective amount or unit dosage of a phorbol ester compound ofFormula I or derivative compound of a phorbol ester of Formula I, whichmay be formulated with one or more pharmaceutically acceptable carriers,excipients, vehicles, emulsifiers, stabilizers, preservatives, buffers,and/or other additives that may enhance stability, delivery, absorption,half-life, efficacy, pharmacokinetics, and/or pharmacodynamics, reduceadverse side effects, or provide other advantages for pharmaceuticaluse. Effective amounts of a phorbol ester compound or related orderivative compound of Formula I (e.g., a unit dose comprising aneffective concentration/amount of TPA, or of a selected pharmaceuticallyacceptable salt, isomer, enantiomer, solvate, polymorph and/or prodrugof TPA) will be readily determined by those of ordinary skill in theart, depending on clinical and patient-specific factors. Suitableeffective unit dosage amounts of the active compounds for administrationto mammalian subjects, including humans, may range from about 10 toabout 1500 μg, about 20 to about 1000 μg, about 25 to about 750 μg,about 50 to about 500 μg, about 150 to about 500 μg, about 125 μg toabout 500 μg, about 180 to about 500 μg, about 190 to about 500 μg,about 220 to about 500 μg, about 240 to about 500 μg, about 260 to about500 μg, about 290 to about 500 μg. In certain embodiments, the diseasetreating effective dosage of a phorbol ester compound or related orderivative compound of Formula I may be selected within narrower rangesof, for example, 10 to 25 μg, 30-50 μg, 75 to 100 μg, 100 to 300 μg, or150 to 500 μg. These and other effective unit dosage amounts may beadministered in a single dose, or in the form of multiple daily, weeklyor monthly doses, for example in a dosing regimen comprising from 1 to5, or 2 to 3, doses administered per day, per week, or per month. In oneexemplary embodiment, dosages of 10 to 30 μg, 30 to 50 μg, 50 to 100 μg,100 to 300 μg, or 300 to 500 μg, are administered one, two, three, four,or five times per day. In more detailed embodiments, dosages of 50-100μg, 100-300 μg, 300-400 μg, or 400-600 μg are administered once or twicedaily. In a further embodiment, dosages of 50-100 μg, 100-300 μg,300-400 μg, or 400-600 μg are administered every other day. In alternateembodiments, dosages are calculated based on body weight, and may beadministered, for example, in amounts from about 0.5 μg/m² to about 300μg/m² per day, about 1 μg/m² to about 200 μg/m², about 1 μg/m² to about187.5 μg/m² per day, about 1 μg/m² per day to about 175 μg/m² per day,about 1 μg/m² per day to about 157 μg/m² per day about 1 μg/m² to about125 μg/m² per day, about 1 μg/m² to about 75 μg/m² per day, 1 μg/m² toabout 50/μg/m² per day, 2 μg/m² to about 50 μg/m² per day, 2 μg/m² toabout 30 μg/m² per day or 3 μg/m² to about 30 μg/m² per day.

In other embodiments, dosages may be administered less frequently, forexample, 0.5 μg/m² to about 300 μg/m² every other day, about 1 μg/m² toabout 200 μg/m², about 1 μg/m² to about 187.5 μg/m² every other day,about 1 μg/m² to about 175 μg/m² every other day, about 1 μg/m² per dayto about 157 μg/m² every other day about 1 μg/m² to about 125 μg/m²every other day, about 1 μg/m² to about 75 μg/m² every other day, 1μg/m² to about 50 μg/m² every other day, 2 μg/m² to about 50 μg/m² everyother day, 2 μg/m² to about 30 μg/m² per day or 3 μg/m² to about 30μg/m² per day. In additional embodiments, dosages may be administered 3times/week, 4 times/week, 5 times/week, only on weekdays, only inconcert with other treatment regimens, on consecutive days, or in anyappropriate dosage regimen depending on clinical and patient-specificfactors.

The amount, timing and mode of delivery of compositions of the inventioncomprising an (alternatively “stroke treating,” “anti-clotting,”“anticholesterolemic,” “vasodilating,” “antihypertensive,” “arteriolarresistance lowering,” “venous capacity increasing,” “heart oxygen demandreducing,” “heart rate decreasing,” “heart rate stabilizing,” “ERKphorsphorylation inducing,” “IL-2 modulating” and/or “neuroprotective”)effective amount of a phorbol ester compound of Formula I or derivativecompound of a phorbol ester of Formula I, will be routinely adjusted onan individual basis, depending on such factors as weight, age, gender,and condition of the individual, the acuteness of the disease and/orrelated symptoms, whether the administration is prophylactic ortherapeutic, and on the basis of other factors known to effect drugdelivery, absorption, pharmacokinetics, including half-life, andefficacy.

An effective dose or multi-dose treatment regimen for the instantdisease treating (alternatively, “stroke treating,” “anti-clotting,”“anticholesterolemic,” “vasodilating,” “antihypertensive,” “ERKphorsphorylation inducing,” “arteriolar resistance lowering,” “venouscapacity increasing,” “heart oxygen demand reducing,” “heart ratedecreasing,” “heart rate stabilizing,” “blood clot decreasing,”“neuroprotective,” “IL-2 modulating” or “NFκB modulating”) formulationsof the invention will ordinarily be selected to approximate a minimaldosing regimen that is necessary and sufficient to substantially preventor alleviate the symptoms of stroke in the subject. A dosage andadministration protocol will often include repeated dosing therapy overa course of several days or even one or more weeks or years. Aneffective treatment regime may also involve prophylactic dosageadministered on a day or multi-dose per day basis lasting over thecourse of days, weeks, months or even years.

Effectiveness of the compositions and methods of the invention in thetreatment of stroke may be demonstrated using a variety of model systemsincluding temporary middle cerebral artery occlusion as shown in Example9, permanent middle cerebral artery occlusion as shown in Example 8,endovascular filament middle cerebral artery occlusion, embolic middlecerebral artery occlusion as shown in Example 7, endothelin-1-inducedconstriction of arteries and veins, or cerebrocortical photothrombosis.Use of the phorbol ester compositions of the present invention willdecrease the symptoms or long term effects exhibited by the modelsystems by 0%, 20%, 30%, 50% or more, up to a 75-90%, 96% or greaterdecrease over control animals.

Effectiveness of the compositions and methods of the invention in thetreatment of stroke may further be demonstrated by a decrease in thesymptoms exhibited in individuals who have suffered a stroke. Suchsymptoms include, but are not limited to, paralysis, spatial impairment,impaired judgment, left-sided neglect, memory loss, aphasia,coordination and balance problems, nausea, vomiting, cognitiveimpairment, perception impairment, orientation impairment, homonymoushemianopsia and impulsivity. Use of the phorbol ester compositions ofthe present invention will decrease the symptoms exhibited byindividuals by 0%, 20%, 30%, 50% or more, up to a 75-90%, 96% or greaterdecrease over initial states.

Within additional aspects of the invention, combinatorial diseasetreating (“stroke treating,” “anti-clotting,” “anticholesterolemic,”“vasodilating,” “antihypertensive,” “ERK phorsphorylation inducing,”“arteriolar resistance lowering,” “venous capacity increasing,” “heartoxygen demand reducing,” “heart rate decreasing,” “heart ratestabilizing,” or “NFκB modulating”) formulations and coordinateadministration methods are provided which employ an effective amount ofa phorbol ester compound of Formula I or a derivative compound ofFormula I and one or more secondary or adjunctive agent(s) that is/arecombinatorially formulated or coordinately administered with the phorbolester compound of Formula I to yield a combined, multi-active diseasetreating composition or coordinate treatment method.

Exemplary combinatorial formulations and coordinate treatment methods inthe prevention or treatment of stoke employ the phorbol ester compoundof Formula I, or derivative compound of a phorbol ester of Formula I, incombination with one or more additional, neuroprotective or otherindicated, secondary or adjunctive therapeutic agents that is/are usefulfor treatment or prophylaxis of the targeted disease, condition and/orsymptom(s). For most combinatorial formulations and coordinate treatmentmethods of the invention, a phorbol ester compound of Formula I orrelated or derivative compound is formulated, or coordinatelyadministered, in combination with one or more secondary or adjunctivetherapeutic agent(s), to yield a combined formulation or coordinatetreatment method that is combinatorially effective or coordinatelyuseful to prevent or treat stroke, or the effects of stroke. Exemplarycombinatorial formulations and coordinate treatment methods in thiscontext employ a phorbol ester compound of Formula I, or derivativecompound of a phorbol ester of Formula I, in combination with one ormore secondary or adjunctive therapeutic agents selected from tissueplasminogen activator, an anticoagulant, a statin, angiotensin IIreceptor blockers, angiotensin-converting enzyme inhibitor,anti-platelet agent, fibrate, beta-blocker, calcium channel blocker, ordiuretic. Exemplary anticoagulants include, but are not limited to,heparin, warfarin, heparinoids, phenindione, atomentin, acenocoumarol,phenprocoumon, idraparinux, fondaparinux, and thrombin inhibitors.Exemplary statins include, but are not limited to, lovastatin,amlodipine, atorvastatin, rosuvastatin, simvastatin, fluvastatin,pitavastatin, and pravastatin. Exemplary angiotensin II receptorblockers include, but are not limited to, candesartan, eprosartan,irbesartan, losartan, olmesartan, telmisartan, and valsartan.Angiotensin converting enzyme inhibitors include, but are not limitedto, enazepril, captopril, enalapril, fosinopril, isinopril, moexipril,perindopril, quinapril, ramipril, and trandolapril. Exemplarybeta-blockers include, but are not limited to, alprenolol, bucindolol,carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol,pindolol propranolol, sotalol, timolol, eucommia, acebutolol, atenolol,betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, and nebivolol.Exemplary calcium channel blockers include, but are not limited to,amlodipine, clevidipine, diltiazem, felodipine, isradipine, nifedipine,nicardipine, nimodipine, nisoldipine, and verapamil. Exemplary diureticsinclude, but are not limited to, chlorothiazide, hydrochlorothiazide,bumetanide, ethacrynic acid, furosemide, amiloride, eplerenone,spironolactone and triamterene. Exemplary fibrates include, but are notlimited to, benzafibrate, ciprofibrate, clofibrate, gemfibrozil orfenofibrate. Exemplary anti-platelet agents include, but are not limitedto, clopidogrel and ticlopidin.

Coordinate treatment methods may further employ surgical interventionincluding, but not limited to, the use of pacemakers, implantabledefibrillators, coronary stents, prosthetic valves, coronary arterybypass, balloon angioplasty, valve repair and replacement, carotidendarterectomy, angioplasty, stent placement, craniotomy, endovascularcoil emobilization, patent foramen ovale closure and hearttransplantation.

In certain embodiments the invention provides combinatorial diseasetreating (“stroke treating,” “anti-clotting,” “anticholesterolemic,”“vasodilating,” “antihypertensive,” “ERK phorsphorylation inducing,”“arteriolar resistance lowering,” “venous capacity increasing,” “heartoxygen demand reducing,” “heart rate decreasing,” “heart ratestabilizing,” or “NFκB modulating”) formulations comprising a phorbolester and one or more adjunctive agent(s) having disease treatingactivity. Within such combinatorial formulations, a phorbol ester ofFormula I and the adjunctive agent(s) having disease treating activitywill be present in a combined formulation in disease treating (“stroketreating,” “anti-clotting,” “anticholesterolemic,” “vasodilating,”“antihypertensive,” “ERK phorsphorylation inducing,” “arteriolarresistance lowering,” “venous capacity increasing,” “heart oxygen demandreducing,” “heart rate decreasing,” “heart rate stabilizing,” or “NFκBmodulating”) effective amounts, alone or in combination. In exemplaryembodiments, a phorbol ester compound of Formula I and a non-phorbolester agent(s) will each be present in a disease treating/preventingamount (i.e., in singular dosage which will alone elicit a detectablealleviation of symptoms in the subject). Alternatively, thecombinatorial formulation may comprise one or both the phorbol estercompound of Formula I and the non-phorbol ester agents insub-therapeutic singular dosage amount(s), wherein the combinatorialformulation comprising both agents features a combined dosage of bothagents that is collectively effective in eliciting a disease, condition,or symptom alleviating response. Thus, one or both of the phorbol esterof Formula I, or derivative compound of a phorbol ester of Formula I,and non-phorbol ester agents may be present in the formulation, oradministered in a coordinate administration protocol, at asub-therapeutic dose, but collectively in the formulation or method theyelicit a detectable decrease in symptoms of disease, the occurrence orrecurrence of stroke, or sequelae from a stroke in the subject. In yetanother embodiment, the combinatorial formulation may include one ormore neuroprotective agents. In a further embodiment, the combinatorialformulation may include one or more anti-inflammatory agents or othersecondary or additional therapeutic agents as described herein.

To practice coordinate administration methods of the invention, aphorbol ester compound of Formula I, or derivative compound of a phorbolester of Formula I, may be administered, simultaneously or sequentially,in a coordinate treatment protocol with one or more of the secondary oradjunctive therapeutic agents contemplated herein. Thus, in certainembodiments a compound is administered coordinately with a non-phorbolester agent, or any other secondary or adjunctive therapeutic agentcontemplated herein, using separate formulations or a combinatorialformulation as described above (i.e., comprising both a phorbol estercompound of Formula I or related or derivative compound, and anon-phorbol ester therapeutic agent). This coordinate administration maybe done simultaneously or sequentially in either order, and there may bea time period while only one or both (or all) active therapeutic agentsindividually and/or collectively exert their biological activities.

In another embodiment, such coordinate treatment methods may, forexample, follow or be derived from various protocols for the treatmentof stroke. Coordinate treatment methods may, for example, include aphorbol ester and/or treatments for prevention or treatment of damagecaused by a stroke. A distinguishing aspect of all such coordinatetreatment methods is that the phorbol ester compound of Formula I orderivative compound of a phorbol ester of Formula I, exerts at leastsome activity, which yields a favorable clinical response in conjunctionwith a complementary stroke preventing or treating agent, or distinct,clinical response provided by the secondary or adjunctive therapeuticagent. Often, the coordinate administration of the phorbol estercompound of Formula I, or derivative compound of a phorbol ester ofFormula I, with the secondary or adjunctive therapeutic agent will yieldimproved therapeutic or prophylactic results in the subject beyond atherapeutic effect elicited by the phorbol ester compound of Formula Ior derivative compound of a phorbol ester of Formula I, or the secondaryor adjunctive therapeutic agent administered alone. This qualificationcontemplates both direct effects as well as indirect effects.

Within exemplary embodiments, a phorbol ester compound of Formula I, orderivative compound of a phorbol ester of Formula I, will becoordinately administered (simultaneously or sequentially, in combinedor separate formulation(s)), with one or more secondary stroke treatingcompounds or other indicated or adjunctive therapeutic agents, e.g.tissue plasminogen activator, an anticoagulant, a statin, angiotensin IIreceptor blockers, angiotensin-converting enzyme inhibitor, fibrate,beta-blocker, calcium channel blocker, lipid-lowering medication,anti-platelet agent or diuretic. In addition, adjunctive or secondarytherapies may be used in the treatment of stroke or the effects ofstroke such as, but not limited to, pacemakers, implantabledefibrillators, coronary stents, prosthetic valves, artificial hearts,coronary artery bypass, balloon angioplasty, valve repair andreplacement, heart transplantation, carotid endarterectomy, angioplasty,stent placement, craniotomy, endovascular coil emobilization, or patentforamen ovale closure.

As noted above, in all of the various embodiments of the inventioncontemplated herein, the disease treating methods and formulations mayemploy a phorbol ester compound of Formula I in any of a variety offorms, including any one or combination of the subject compound'spharmaceutically acceptable salts, solvates, isomers, enantiomers,polymorphs, solvates, hydrates, and/or prodrugs. In exemplaryembodiments of the invention, TPA is employed within the therapeuticformulations and methods for illustrative purposes.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended therapeutic orprophylactic purpose. Suitable routes of administration for thecompositions of the invention include, but are not limited to,conventional delivery routes, devices and methods including injectablemethods such as, but not limited to, intravenous, intramuscular,intraperitoneal, intraspinal, intrathecal, intracerebroventricular,intraarterial, subcutaneous and intranasal routes.

The compositions of the present invention may further include apharmaceutically acceptable carrier appropriate for the particular modeof administration being employed. Dosage forms of the compositions ofthe present invention include excipients recognized in the art ofpharmaceutical compounding as being suitable for the preparation ofdosage units as discussed above. Such excipients include, withoutintended limitation, binders, fillers, lubricants, emulsifiers,suspending agents, sweeteners, flavorings, preservatives, buffers,wetting agents, disintegrants, effervescent agents and otherconventional excipients and additives.

If desired, the compositions of the invention can be administered in acontrolled release form by use of a slow release carrier, such as ahydrophilic, slow release polymer. Exemplary controlled release agentsin this context include, but are not limited to, hydroxypropyl methylcellulose, having a viscosity in the range of about 100 cps to about100,000 cps or other biocompatible matrices such as cholesterol.

Some phorbol ester compositions of Formula I of the invention aredesigned for parenteral administration, e.g. to be administeredintravenously, intramuscularly, subcutaneously or intraperitoneally,including aqueous and non-aqueous sterile injectable solutions which,like many other contemplated compositions of the invention, mayoptionally contain anti-oxidants, buffers, bacteriostats and/or soluteswhich render the formulation isotonic with the blood of the mammaliansubject; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and/or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers. Additionalcompositions and formulations of the invention may include polymers forextended release following parenteral administration. The parenteralpreparations may be solutions, dispersions or emulsions suitable forsuch administration. The subject agents may also be formulated intopolymers for extended release following parenteral administration.Pharmaceutically acceptable formulations and ingredients will typicallybe sterile or readily sterilizable, biologically inert, and easilyadministered. Such polymeric materials are well known to those ofordinary skill in the pharmaceutical compounding arts. Parenteralpreparations typically contain buffering agents and preservatives, andinjectable fluids that are pharmaceutically and physiologicallyacceptable such as water, physiological saline, balanced salt solutions,aqueous dextrose, glycerol or the like. Extemporaneous injectionsolutions, emulsions and suspensions may be prepared from sterilepowders, granules and tablets of the kind previously described.Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as described herein above, or an appropriatefraction thereof, of the active ingredient(s).

In more detailed embodiments, compositions of the invention may comprisea phorbol ester compound of Formula I or derivative compound of aphorbol ester of Formula I encapsulated for delivery in microcapsules,microparticles, or microspheres, prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively; in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules); or withinmacroemulsions.

As noted above, in certain embodiments the methods and compositions ofthe invention may employ pharmaceutically acceptable salts, e.g., acidaddition or base salts of the above-described phorbol ester compounds ofFormula I and/or related or derivative compounds. Examples ofpharmaceutically acceptable addition salts include inorganic and organicacid addition salts. Suitable acid addition salts are formed from acidswhich form non-toxic salts, for example, hydrochloride, hydrobromide,hydroiodide, sulphate, hydrogen sulphate, nitrate, phosphate, andhydrogen phosphate salts. Additional pharmaceutically acceptable saltsinclude, but are not limited to, metal salts such as sodium salts,potassium salts, cesium salts and the like; alkaline earth metals suchas calcium salts, magnesium salts and the like; organic amine salts suchas triethylamine salts, pyridine salts, picoline salts, ethanolaminesalts, triethanolamine salts, dicyclohexylamine salts,N,N′-dibenzylethylenediamine salts and the like; organic acid salts suchas acetate, citrate, lactate, succinate, tartrate, maleate, fumarate,mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, andformate salts; sulfonates such as methanesulfonate, benzenesulfonate,and p-toluenesulfonate salts; and amino acid salts such as arginate,asparginate, glutamate, tartrate, and gluconate salts. Suitable basesalts are formed from bases that form non-toxic salts, for examplealuminum, calcium, lithium, magnesium, potassium, sodium, zinc anddiethanolamine salts.

Other detailed embodiments, the methods and compositions of theinvention for employ prodrugs of phorbol esters of Formula I. Prodrugsare considered to be any covalently bonded carriers which release theactive parent drug in vivo. Examples of prodrugs useful within theinvention include esters or amides with hydroxyalkyl or aminoalkyl as asubstituent, and these may be prepared by reacting such compounds asdescribed above with anhydrides such as succinic anhydride.

The invention disclosed herein will also be understood to encompassmethods and compositions comprising phorbol esters of Formula I using invivo metabolic products of the said compounds (either generated in vivoafter administration of the subject precursor compound, or directlyadministered in the form of the metabolic product itself). Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification and the like of the administered compound,primarily due to enzymatic processes. Accordingly, the inventionincludes methods and compositions of the invention employing compoundsproduced by a process comprising contacting a phorbol ester compound ofFormula I with a mammalian subject for a period of time sufficient toyield a metabolic product thereof. Such products typically areidentified by preparing a radiolabelled compound of the invention,administering it parenterally in a detectable dose to an animal such asrat, mouse, guinea pig, monkey, or to man, allowing sufficient time formetabolism to occur and isolating its conversion products from theurine, blood or other biological samples.

The invention disclosed herein will also be understood to encompassdiagnostic compositions for diagnosing the risk level, presence,severity, or treatment indicia of, or otherwise managing diseasesincluding, but not limited to, stroke, in a mammalian subject,comprising contacting a labeled (e.g., isotopically labeled, fluorescentlabeled or otherwise labeled to permit detection of the labeled compoundusing conventional methods) phorbol ester compound of Formula I to amammalian subject (e.g., to a cell, tissue, organ, or individual) atrisk or presenting with one or more symptom(s) of stroke, and thereafterdetecting the presence, location, metabolism, and/or binding state ofthe labeled compound using any of a broad array of known assays andlabeling/detection methods. In exemplary embodiments, a phorbol estercompound of Formula I is isotopically-labeled by having one or moreatoms replaced by an atom having a different atomic mass or mass number.Examples of isotopes that can be incorporated into the disclosedcompounds include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively. Theisotopically-labeled compound is then administered to an individual orother subject and subsequently detected as described above, yieldinguseful diagnostic and/or therapeutic management data, according toconventional techniques.

EXAMPLES

The experiments described below demonstrate novel and powerful uses forphorbol esters and derivative compounds as stroke treating andpreventing agents. These and additional findings are further expandedand elucidated within the following examples.

Example I Effect of TPA on the Peripheral White Blood Cells (WBC) andHemoglobin (Hb) Counts in S180 Cell-Injected Mice

Sarcoma 180 (S180) cells were injected into Kwen-Ming mice. On the thirdday, the mice were given TPA interperitoneally (i.p.). at 50, 100 or 200μg/kg/day for 7 days. On the second day after the treatment wascompleted, blood samples were taken from the tails of the treated micefor WBC and Hb analyses. The WBC counts for the treated groups (50, 100,or 200 ug/kg/day for 7 days) were 16.1±7.4, 18.7±0.3.0 and20.7±0.3.4×10⁹/L, respectively; the WBC count for the control group was13.6±1.8×10⁹/L. The Hb of the treated groups were 136±11, 149±12 and149±10 g/L, and the Hb of the control group was 134+−15 g/L. The resultsindicate that i.p. injection of TPA could increase the peripheral WBCcounts in mice in a dose-dependent manner, whereas the Hb levels werenot greatly affected in TPA treated mice when compared to the controlmice.

Example II Dose Ranging Study

Due to the strong local irritation caused by TPA application, TPA wasgiven to patients by intravenous (i.v.) infusion. TPA solution in asterile syringe was injected into 200 ml of sterile saline and mixedwell for i.v. infusion.

The Toxicity and Side Effects of Different TPA Doses AdministeredClinically:

(1) TPA Given at 1 mg/Patient/Week:

One mg TPA in solution was mixed well with 200 ml of sterile saline forintravenous infusion which was completed in 1 h at the rate of 16μg/min. One hour after TPA administration, patients started to havechills which lasted for about 30 min, followed by fever, (the patients'temperature reached 37.5-39.5° C. which lasted for 3-5 h, then returnedto normal) with light to heavy perspiration. The above symptoms could bealleviated by giving the patients glucocorticoids. TPA at this dosecaused a minority of patients to bleed, several patients suffered for ashort period of time difficulty in breathing, and Hb was detected in theurine. However, these side effects were short lived and reversible. Thecardiac, hepatic, renal and pulmonary functions were all found to benormal.

(2) TPA Given at 0.5 mg/Patient×2/Week: (Two Doses a Week)

0.5 mg of TPA in solution was mixed well with 200 ml of saline forintravenous infusion which was completed in 1 h at the rate of 8 μg/min.The reactions after administration were similar to that of the 1 mg TPAdosage, but to a lesser extent than the 1 mg dose. The patientstolerated the lower dose more easily. Occasionally, Hb was detected inpatients' urine. Difficulty in breathing was not observed. The cardiac,hepatic, renal and pulmonary functions were all normal.

(3) TPA Given at 0.25 mg/Patient×4/Week:

0.25 mg of TPA in solution was mixed well with 200 ml of saline forintravenous infusion which was completed in 1 h at the rate of 4 μg/min.After administration, symptoms such as chills and fever were alsoobserved, but to a much lesser extent than with the higher dosages. NoHb was detected in the urine, and no patient suffered difficulty inbreathing. The cardiac, hepatic, renal and pulmonary functions were allnormal.

Example III Treatment of Relapsed/Refractory Malignancies with TPA

Patients with histologically documented relapsed/refractory hematologicmalignancy/bone marrow disorders are treated with a combination of TPA(Xichuan Pharmaceuticals, Nan Yang, Henan, China), dexamethasone andcholine magnesium trisalicylate. Comparable methods as set forth belowfor demonstrating the therapeutic use of TPA in the treatment of AcuteMyelogenous Leukemia (AML) will be applied to demonstrate the use of TPAfor treating other neoplastic conditions and malignancies. In additionto the specific protocols herein, successful treatment and/or remissionwill be determined for different targeted neoplastic and malignantconditions using any of a wide variety of well known cancer detectionand assessment methods—for example by determining size reduction ofsolid tumors, histopathological studies to evaluate tumor growth, stage,metastatic potential, presence/expression levels of histological cancermarkers, etc.

AML is an aggressive disease that generally warrants urgent andintensive therapy. The average patient age at AML diagnosis is 64-68years old, and patients over the age of 60 treated with standardchemotherapy are cured of their disease <20% of the time. Patients whodevelop AML after an antecedent hematologic disorder or priorleukemogenic chemotherapy/radiation therapy have similarly pooroutcomes, as do patients whose disease is associated with specificadverse cytogenetic and clinical features. Hence, most patientsdiagnosed with AML have patient and/or disease-related features that areassociated with a very poor prognosis. For patients with relapseddisease, no standard non-transplant therapy has demonstrated thecapacity for cure. For these patients, AML is often a fatal disease. Newapproaches to the therapy of AML are needed.

Employing the methods and compositions of the instant invention, TPA, isdeveloped as a therapeutic agent for treating patients with AML, basedon TPA's novel role in modulating intracellular signaling pathways, it'scapacity to induce differentiation and/or apoptosis in cell lines, andclinical data indicating the effectiveness of TPA in treating neoplasticand malignant disorders, including myeloid malignancies.

Thus far clinical evaluation of TPA has demonstrated that TPA exertsdirect therapeutic cytotoxic effects in at least a subset of AML cases,as measured by cell viability and apoptosis assays. In all primarycultures analyzed by Western analysis, TPA strongly induced ERKphosphorylation by 1 hour in culture. TPA's cytotoxic effect on primaryAML cells is associated with the subsequent loss of the phospho-ERKpro-survival signal after 24 hour ex vivo exposure. This observation isin good agreement with other studies that reported decreased primary AMLsurvival after pharmacological interruption of ERK signaling by MEKinhibitors, such as PD98059, U0126 and PD 184352. In our studies, lossof ERK signaling was associated with induction of ERK phosphatases.

In addition to protein kinase C and ERK activation, TPA is a knowninducer of NF-κB, a pro-survival transcription factor oftenconstitutively active in AML blasts and leukemic stem cells. Recent workfrom our laboratory has demonstrated that AML cell NF-κB can beinhibited in vivo with 48 h of treatment with dexamethasone+cholinemagnesium trisalicylate (CMT). In addition, we have shown thatdexamethasone can induce MKP-1 ERK phosphatase expression and enhanceTPA cytotoxicity on primary AML samples. In this context, we have chosenin exemplary embodiments below to use dexamethasone and CMT asadjunctive medications to be used 24 h pre- and 24 h post treatment withTPA. These medications are well-tolerated and anticipated to reduceinflammatory adverse effects of treatment and enhance TPA cytotoxicityby increasing ERK phosphatase expression and inhibiting NF-κB. Inaddition dexamethasone and CMT will be used as adjunctive medicationsbecause they are anti-inflammatory, may ameliorate adverse effects, andmay enhance anti-leukemic activity by inhibition of the anti-apoptoticeffects of constitutive NF-κB expression and induction of phosphatasesthat decrease signaling pathway activity.

An initial TPA Phase 1 study enrolled 35 patients [23 withrelapsed/refractory AML, 2 with other myeloid malignancies (CML-blastcrisis, myelodysplasia with excess blasts), 3 with Hodgkin's Disease, 3with non-Hodgkin's lymphoma and 4 with solid tumors]. The majority ofpatients had relapsed/refractory AML. Our clinical results include oneAML patient with stable disease for >5 months, who received 8 TPAinfusions. In a second AML patient, a pronounced (5-fold) decline in thenumber of circulating blasts was seen following TPA administration. Thisdecline in leukemic blasts persisted for 4 weeks, and the patienteventually died from a fungal infection. Finally, a patient withrelapsed and refractory Hodgkin's disease despite high dose chemotherapywith autologous stem cell rescue had a partial remission of a chest wallmass after TPA administration. TPA dose escalation has been completed,in the last cohort 2 out of 3 patients treated at a dose of 0.188 mg/m2d1-5, 8-12 experienced grade III non-hematologic dose limitingtoxicities (DLT), establishing the maximum tolerated TPA dose as asingle agent at 0.125 mg/m2/d on d1-5 and 8-12.

In the case of AML and other hematologic malignancies, patients aregiven an initial dose of TPA of 1 mg/week×3 weeks (days 1, 8, 15)administered with continuous/intermittent pulse oximetry for 6 hours.Twenty four hours prior to initiation of TPA therapy, patients are given10 mg of dexamethasone every six hours and 1500 mg of choline magnesiumtrisalicylate (CMT) every eight hours continuing until 24 hours afteradministration of TPA. After administration of the initial dose of TPA,patients have a two week rest period after which they may bereevaluated. Those patients that have a disease response orstabilization from the initial dose of TPA are treated for up to sixcycles of twenty-eight days according to the protocol below.

Following the two week rest period, patients are pre-medicated withTylenol 650 mg and Benadryl 25-50 mg (depending on the patient's sizeand age) thirty minutes prior to administration of TPA. They are thengiven an intravenous infusion of TPA through a central venous catheterdaily for 5 days a week for two consecutive weeks followed by a 2-weekrest period. TPA is administered at a dose of 1 mg in 200 ml of normalsaline over 1 hour. Twenty four hours prior to initiation of TPAtherapy, patients are given 10 mg of dexamethasone every six hours and1500 mg of choline magnesium trisalicylate continuing every eight hoursuntil 24 hours after administration of the TPA.

Blood levels of TPA are measured prior to and after infusion using abioassay that measures organic solvent extractable differentiationactivity. 1 ml of blood is extracted twice with 5 ml of ethyl acetate,redissolving the extraction residue in 50 μL of ethanol and addition ofan aliquot of HL60 cells. After 48 hours, adherent cells are measured.

Tests are also run on blood samples taken prior to and after infusionwith TPA to determine levels of white blood cells, platelets, andneutrophils. The samples are additionally analyzed for the presence ofmyeloblasts and Auer rods. These and continuing experiments will furtherelucidate the therapeutic cytotoxic and other effects that TPA elicitsagainst neoplastic cells in AML and other neoplastic and malignantconditions.

Example IV Measurement of the Modulation of ERK Activation

Phospho-ERK levels are measured in circulating malignant cells inpatients with leukemia and in peripheral blood mononuclear cells inlymphoma/solid tumor patients. A blood sample is taken from patientstreated according to the protocol of Example III both prior to and afteradministration of TPA.

In leukemia patients with a WBC≥1000 per μL, flow cytometry is performedon a blood sample using cell surface antigen-specific and phospho-ERKspecific antibodies directly conjugated to flurophores (BD Biosciences,San Jose, Calif.). Samples are taken pre-administration of TPA and onehour after infusion of TPA on days 1, 2, and 11 in the initial treatmentaccording to the protocol of Example III and days 1 and 11 in subsequentcycles. In leukemia patients with an absolute leukemic blast number≥2500 per μL and other non-leukemic patients, peripheral blood samplesare taken on days 1, 8 and 15 of the initial cycle according to theprotocol of Example III prior to and 1 and 4 hours post infusion.Samples are also analyzed using Western blot analysis for phosphor-ERK,and total ERK1/2 levels to confirm the results obtained from the flowcytometry and correlated to clinical responses.

The foregoing analyses will further elucidate TPA's role in treatment ofneoplastic and malignant conditions, including TPA's cytotoxic effect onmalignant cells, exemplified by primary AML cells, and the associatedreduction by TPA of the phosphor-ERK pro-survival signal.

Example V Measurement of NF-κB Modulation

In prior studies we have shown that NF-κB activity can be modulated inpatients following administration of TPA with dexamethasone.Additionally, dexamethasone has been shown to induce MKP-1 ERKphosphatase expression and enhance TPA cytotoxicity. The followingstudies are designed to further elucidate how NF-κB activity istherapeutically modulated in patients treated with TPA plusdexamethasone.

NF-κB binding is measured in patient peripheral blood samples atbaseline and pre and post infusion from patients treated with TPAaccording to Example III using ELISA-based assays (BD Bioscience, SanJose, USA). NF-κB levels are quantified using chemiluminescent intensityto detect binging in limiting amounts of cellular extract using a96-well format. Additionally, electrophoretic mobility shift assays areperformed to measure NF-κB binding in peripheral blood samples fromleukemia patient with an absolute leukemic blast number ≥2500 per μL andother non-leukemic patients with normal white blood cell counts.

The foregoing studies will further show that TPA is an inducer of NF-κB;however these experiments demonstrate that AML cell NF-κB can beinhibited with treatment with dexamethasone and choline magnesiumtrisalicylate.

Example VI Treatment of Individuals Who have Suffered a Stroke

Patient N.C., male, 68, suffered a stroke eighteen months prior totreatment with TPA. At the time TPA treatment was initiated, he wasunable to walk without a cane, had difficulty with both his left handand left leg and was tired and weak. He received injections of 1 ampoulecontaining 0.19 mg of TPA (0.125 mg/m²) every other day for four weeks,then 0.24 mg of TPA (1.25×0.125 mg/m²) every other day for 2 weeks, andthen 0.26 mg of TPA (1.5×0.125 mg/m²) every other day for an additional3 weeks. The patient has recovered fully.

Patient M.C., male, age 65, suffered a stroke seven years prior tobeginning treatment with TPA. He received 3-4 injections of 0.19 mg ofTPA (0.125 mg/m²) per week for ten weeks for a total of 35 injections.He has regained mobility in his face and had an 80% improvement in themobility of his right side.

Example VII Treatment of Embolic Stroke Model with TPA

Male Sprague-Dawley rats (Charles River, Japan) each having a bodyweight of 280-350 g are used. An embolic stroke is induced following amodification of the method of Kudo, et al. (1982) The rats to be usedfor the collection of blood are anesthetized with 1.0% halothane(Fluorothane™; Takeda, Osaka, Japan) under spontaneous respiration. A24-gauge Surflo™ (Terumo Medical Products, Elkton, Md.) is secured inthe femoral artery and 0.1 mL of arterial blood is taken with a 1-mLsyringe for injection (Terumo Medical Products, Elkton, Md.). The arteryblood in the syringe is incubated at 30° C. for 2 days to form a bloodclot. After that, 0.1 mL of physiological saline is added to the syringefor injection and passed through a 26-gauge injection needle (TerumoMedical Products, Elkton, Md.) twice so that the blood clot is crushed.

Rats in which a cerebral embolic stroke is induced are anesthetized with1.0% halothane under spontaneous respiration. The neck of the rats issubjected to a midline incision and external carotid artery, superiorthyroid artery, occipital artery and pterygopalatine artery arecauterized with a bipolar coagulator (T-45; Keisei Medical IndustrialCo. Ltd, Tokyo, Japan). Cerebral embolism is induced by injecting 0.1 mLof the crushed blood clot into the internal carotid.

Evaluation of the formation of a cerebral embolism is carried out usinga laser Doppler flowmetry (FloC1; Omegawave, Tokyo, Japan). A decreasein cerebral blood flow to a level of 30% or less is taken as a positiveevidence of embolism formation. The cerebral blood flow is monitored for30 minutes after infusion of the blood clot and blood flow is monitoredas remaining at 50% or less of the flow prior to the injection of theblood clot. After that, a cannula (PESO) for administration of themedicament is secured in the jugular vein and the animals are woken.

The rats that have successfully formed a cerebral embolism are dividedinto four groups. The first group of rats is given a saline injectionevery other day. Groups 2-4 are given 0.125 mg/m² injection of TPA everyother day for four weeks. Group 2 is then sacrificed. Groups 3-4 aregiven a further 0.156 mg/m² of TPA every other day for two weeks andthen Group 3 is sacrificed. Group 4 is given 0.18775 mg/m² of TPA everyother day for three weeks and then sacrificed.

The brains are excised after the animals are sacrificed and sliced inten sections at 1 mm intervals using a McIwain tissue chopper (MickleLaboratory Engineering, U.K.) and are stained by dipping for 20 minutesin a 2% TTC (2,3,5-triphenyltetrazolium chloride; Tokyo Kasei) at 37° C.Images of the TTC-stained slices are uploaded into a computer using adigital camera (HC-2500; Fuji PhotoFilm) and Phatograb-2500 (Fuji PhotoFilm) and infarct volume is calculated using Mac Scope (Mitani, Japan).Infarct volume is given by a mean value±standard error. With regard tothe statistical test of the result of the infarct volume, the evaluationis done by carrying out a Dunnett's test for control group and for eachof the TPA-administered groups as compared with the control group andthen by carrying out the t-test for the TPA-administered group.

Neurological symptoms are observed daily until sacrifice and the ratsare evaluated according to three tests: (1) Rats are held gently by thetail, suspended one meter above the floor, and observed for forelimbflexion; (2) Rats are placed on a large sheet of soft, plastic coatedpaper that could be gripped firmly by their claws. With the tail held byhand, gentle lateral pressure is applied behind the rat's shoulder untilthe forelimbs slid several inches; (3) Rats are allowed to move aboutfreely and are observed for circling behavior. Scoring of theneurological symptoms is carried out according to the scale developed byBederson et al. (1986) as follows: 0: no observable deficit; 1: forelimbflexion; 2: decreased resistance to lateral push without circling; 3:same behavior as grade 2, with circling.

Neurological symptoms are evaluated using a Steel's test for the controlgroup and for each of the TPA administered groups as compared with thecontrol group and then by carrying out a Wilcoxon test for the TPAadministered group. In any of the tests, the value where p<0.05 isdefined to be statistically significant.

Example VIII Effectiveness of TPA in the Treatment of Stroke Using aPermanent Middle Cerebral Artery Occlusion Model

Male Wistar rats (250-320 g) are used for this study. Animals areanesthetized with Isoflurane (3% induction, 1-2% maintenance).Anesthesia is monitored by toe pinch. Aseptic technique is used for allprocedures during this study. The surgical site is clipped and cleanedwith alcohol and surgical scrub. The animal is placed on a warm waterheating pad to maintain body temperature. A paramedian incision is madeon the neck over the carotid artery. The tissue is bluntly dissectedaway to reveal the carotid artery and the bifurcation. Sutures areplaced around the proximal portion or the common carotid and theexternal carotid arteries. These sutures are tied off. An incision ismade in the common carotid, distal to the ligation. A pre-preparedfilament (4-0 monofilament suture or like material) is placed in thecarotid and advanced into the internal carotid artery. The filament isadvanced about 20 mm past the carotid bifurcation until slightresistance is felt as it wedges in the middle cerebral artery. Care mustbe taken to not rupture the artery upon insertion of the filament. Thefilament is tied in place and the skin incision closed. The animal isevaluated when awake for successful occlusion using the Bederson scale.(See Bederson et al., (1986) Stroke, 17:1304-1308.) Body temperature istaken every 15 minutes to maintain normothermia. Animals that haveundergone the middle cerebral artery occlusion procedure may havedifficulty in thermoregulation for a few hours after surgery Animals areplaced in a cooling or heating box as determined by their temperature.Body temperature is maintained at 37.5° C. Animals are monitored for 6hours following middle cerebral artery and are then placed in cagesovernight.

The rats are divided into four groups. The first group of rats is givensaline injections every other day. Groups 2-4 are given 0.125 mg/m²injection of TPA every other day for four weeks. Group 2 is thensacrificed. Groups 3-4 are given a further 0.156 mg/m² of TPA everyother day for two weeks and then Group 3 is sacrificed. Group 4 is given0.18775 mg/m² of TPA every other day for three weeks and thensacrificed.

The brains are excised after the animals are sacrificed and sliced inten sections at 1 mm intervals using a McIwain tissue chopper (MickleLaboratory Engineering, U.K.) and are stained by dipping for 20 minutesin a 2% TTC (2,3,5-triphenyltetrazolium chloride; Tokyo Kasei) at 37° C.Images of the TTC-stained slices are uploaded into a computer using adigital camera (HC-2500; Fuji PhotoFilm) and Phatograb-2500 (Fuji PhotoFilm). Brain slices are photographed and analyzed for infarct size,infarct volume, penumbra, and edema.

Neurological symptoms are observed daily until sacrifice. Neurologicalsymptoms are observed daily until sacrifice and the rats are evaluatedaccording to three tests. (1) Rats are held gently by the tail,suspended one meter above the floor, and observed for forelimb flexion.(2) Rats are placed on a large sheet of soft, plastic coated paper thatcould be gripped firmly by their claws. With the tail held by hand,gentle lateral pressure is applied behind the rat's shoulder until theforelimbs slid several inches. (3) Rats are allowed to move about freelyand are observed for circling behavior. Scoring of the neurologicalsymptoms is carried out according to the scale developed by Bederson etal. (1986) as follows: 0: no observable deficit; 1: forelimb flexion; 2:decreased resistance to lateral push without circling; 3: same behavioras grade 2, with circling.

Neurological symptoms are evaluated using a Steel's test for the controlgroup and for each of the TPA administered groups as compared with thecontrol group and then by carrying out a Wilcoxon test for the TPAadministered group. In any of the tests, the value where p<0.05 isdefined to be statistically significant.

Example IX Effectiveness of TPA in the Treatment of Stroke Using aTemporary Middle Cerebral Artery Occlusion Model

Male C57B16 mice (25-30 g) are used in this study. Mice are anesthetizedwith Isoflurane (3% induction, 1-2% maintenance). The surgical site isclipped and cleaned with alcohol and surgical scrub. A midline neckincision is made over the carotid artery and the artery is dissected toits bifurcation. A monofilament suture is introduced into the internalcarotid artery and advanced until it lodges in the middle cerebralartery. The suture is tied in placed and the incision is closed. Twohours after occlusion the mice will be re-anesthetized and the suturewill be removed from the MCA. Body temperature is maintained by use of aheating pad both during and after surgery. Animals are monitored for 4hours following middle cerebral artery occlusion.

The rats are divided into four groups. The first group of rats is givensaline injections every other day. Groups 2-4 are given 0.125 mg/m²injection of TPA every other day for four weeks. Group 2 is thensacrificed. Groups 3-4 are given a further 0.156 mg/m² of TPA everyother day for two weeks and then Group 3 is sacrificed. Group 4 is given0.18775 mg/m² of TPA every other day for three weeks and thensacrificed.

The brains are excised after the animals are sacrificed and sliced inten sections at 1 mm intervals using a McIwain tissue chopper (MickleLaboratory Engineering, U.K.) and are stained by dipping for 20 minutesin a 2% TTC (2,3,5-triphenyltetrazolium chloride; Tokyo Kasei) at 37° C.Images of the TTC-stained slices are uploaded into a computer using adigital camera (HC-2500; Fuji PhotoFilm) and Phatograb-2500 (Fuji PhotoFilm). Brain slices are photographed and analyzed for infarct size,infarct volume, penumbra, and edema.

Neurological symptoms are observed daily until sacrifice and the ratsare evaluated according to three tests. (1) Rats are held gently by thetail, suspended one meter above the floor, and observed for forelimbflexion. (2) Rats are placed on a large sheet of soft, plastic coatedpaper that could be gripped firmly by their claws. With the tail held byhand, gentle lateral pressure is applied behind the rat's shoulder untilthe forelimbs slid several inches. (3) Rats are allowed to move aboutfreely and are observed for circling behavior. Scoring of theneurological symptoms is carried out according to the scale developed byBederson et al. (1986) as follows: 0: no observable deficit; 1: forelimbflexion; 2: decreased resistance to lateral push without circling; 3:same behavior as grade 2, with circling.

Neurological symptoms are evaluated using a Steel's test for the controlgroup and for each of the TPA administered groups as compared with thecontrol group and then by carrying out a Wilcoxon test for the TPAadministered group. In any of the tests, the value where p<0.05 isdefined to be statistically significant.

Example X Clinical Effectiveness of the Use of TPA to Treat Stroke

Males and Females between the ages of 30-72 years who suffered a strokeless than one month previously are recruited for participation in a tenweek trial of TPA.

Recruited individuals sign an informed consent form and are evaluatedusing computed tomography (CT), physical and neurological tests,neurological check, sedation level, National Institute of Health StrokeSurvey (NIHSS), 12-lead electrocardiogram, telemetry ofelectrocardiogram, pulse oxygen measurement, vital sign, body weight,background of the patient, test on pregnancy, measurement of medicamentin urine, hematological test, coagulation panel, general clinical test,urine test. Clinical Laboratory Testing includes a Complete MetabolicPanel (Na, K, Cl, CO2, Glu, BUN, Cr, Ca, TP, Alb, TBili, AP, AST, ALT),Hematology CBC (Hgb, Hct, RBC, WBC, Plt, Diff), and Serum hCG for allfemales.

Individuals are administered 0.125 mg/m² of TPA or placebo every otherday for four weeks, then 1.25×0.125 mg/m² or placebo every other day forweeks five and six and 1.5×0.125 mg/m² or placebo every other day forweeks seven to nine. Individuals are monitored during and for two hoursafter administration of TPA or placebo.

At weeks five and week ten, subjects are evaluated using the NIHSS (NIHStroke Scale), the Barthel ADL index (Granger, 1979), and a modifiedRankin scale (Farrell, 1991),

Efficacy is determined by measuring the change from baseline in theNIHSS in individuals treated with TPA in comparison to placebo.Secondary efficacy variables are the Barthel ADL index and a modifiedRankin scale. Safety measures are collected and evaluated through thetrial, specifically measuring changes from baseline visit to week 5.These measures include adverse event reports, physical examinations,vital signs, weight measurements, ECGs, clinical laboratory testresults, and vital signs as well as scores for suicidal behaviors and/orideation. Adverse events are any untoward medical event occurring in asubject administered study drug, irrespective of whether it has a causalrelationship to the study drug. An adverse event can therefore be anyunfavorable or unintended sign (including abnormal laboratory findings,for example), symptom, or disorder temporarily associated with studydrug, whether or not considered related to the study drug.

Subjects are considered to have completed the study if they complete allof the visits. They may be terminated from the study if they fail tomeet inclusion/exclusion criteria; suffer from an adverse event, have aninsufficient therapeutic response, withdraw their consent, violate theprotocol, stop coming, or die.

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We claim:
 1. A method for alleviating or treating impaired mobility asan effect or symptom of stroke in a mammalian subject comprisingadministering an effective amount of phorbol ester of Formula I or apharmaceutically-acceptable salt, isomer, or enantiomer, thereof to saidsubject

wherein R1 and R2 are selected from the group consisting of hydrogen,hydroxyl,

wherein the alkyl group contains 1 to 15 carbon atoms,

wherein the lower alkenyl group contains 1 to 7 carbon atoms,

and R3 is hydrogen or


2. The method of claim 1, wherein R1 or R2 is

the remaining R1 and R2 is

and R3 is hydrogen.
 3. The method of claim 1, wherein the phorbol esteris phorbol 13-butyrate, phorbol 12-decanoate, phorbol 13-decanoate,phorbol 12,13-diacetate, phorbol 13,20-diacetate, phorbol12,13-dibenzoate, phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate,phorbol 12,13-dihexanoate, phorbol 12,13-dipropionate, phorbol12-myristate, phorbol 13-myristate, phorbol 12,13,20-triacetate,12-deoxyphorbol 13-angelate, 12-deoxyphorbol 13-angelate 20-acetate,12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate,12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol13-tetradecanoate, phorbol 12-tigliate 13-decanoate, 12-deoxyphorbol13-acetate, phorbol 12-acetate, or phorbol 13-acetate.
 4. The method ofclaim 1, wherein the phorbol ester is12-O-tetradecanoylphorbol-13-acetate.
 5. The method of claim 1, furthercomprising administering at least one secondary or adjunctivetherapeutic agent that is effective in a combinatorial formulation orcoordinate treatment regimen with said phorbol ester of Formula I. 6.The method of claim 5, wherein the at least one secondary or adjunctivetherapeutic agent is administered to said subject in a coordinateadministration protocol, simultaneously with, prior to, or after,administration of said phorbol ester to said subject.
 7. The method ofclaim 5, wherein the at least one secondary or adjunctive therapeuticagent is tissue plasminogen activator, an anticoagulant, a statin,fibrate, angiotensin II receptor blockers, angiotensin-converting enzymeinhibitor, beta-blocker, anti-platelet agent, calcium channel blocker,or diuretic.
 8. The method of claim 1, further comprising surgicalintervention in combination with phorbol ester of Formula I to treat orprevent effects of stroke in said subject.
 9. The method of claim 8,wherein the surgical intervention is a carotid endarterectomy,angioplasty, stent placement, craniotomy, insertion of a pacemaker,implantation of a defibrillator, replacement of valves, coronary arterybypass, heart transplantation, endovascular coil emobilization, orpatent foramen ovale closure.
 10. The method of claim 1, wherein theimpaired mobility is the effect of stroke.
 11. The method of claim 1,wherein the impaired mobility is the symptom of stroke.